Mixed histiocytic neoplasms: A multicentre series revealing diverse somatic mutations and responses to targeted therapy.

Histiocytic neoplasms are diverse clonal haematopoietic disorders, and clinical disease is mediated by tumorous infiltration as well as uncontrolled systemic inflammation. Individual subtypes include Langerhans cell histiocytosis (LCH), Rosai-Dorfman-Destombes disease (RDD) and Erdheim-Chester disease (ECD), and these have been characterized with respect to clinical phenotypes, driver mutations and treatment paradigms. Less is known about patients with mixed histiocytic neoplasms (MXH), that is two or more coexisting disorders. This international collaboration examined patients with biopsy-proven MXH with respect to component disease subtypes, oncogenic driver mutations and responses to conventional (chemotherapeutic or immunosuppressive) versus targeted (BRAF or MEK inhibitor) therapies. Twenty-seven patients were studied with ECD/LCH (19/27), ECD/RDD (6/27), RDD/LCH (1/27) and ECD/RDD/LCH (1/27). Mutations previously undescribed in MXH were identified, including KRAS, MAP2K2, MAPK3, non-V600-BRAF, RAF1 and a BICD2-BRAF fusion. A repeated-measure generalized estimating equation demonstrated that targeted treatment was statistically significantly (1) more likely to result in a complete response (CR), partial response (PR) or stable disease (SD) (odds ratio [OR]: 17.34, 95% CI: 2.19-137.00, p = 0.007), and (2) less likely to result in progression (OR: 0.08, 95% CI: 0.03-0.23, p < 0.0001). Histiocytic neoplasms represent an entity with underappreciated clinical and molecular diversity, poor responsiveness to conventional therapy and exquisite sensitivity to targeted therapy.

Erdheim-Chester disease: consensus recommendations for evaluation, diagnosis, and treatment in the molecular era.

Erdheim-Chester disease (ECD) is a rare histiocytosis that was recently recognized as a neoplastic disorder owing to the discovery of recurrent activating MAPK (RAS-RAF-MEK-ERK) pathway mutations. Typical findings of ECD include central diabetes insipidus, restrictive pericarditis, perinephric fibrosis, and sclerotic bone lesions. The histopathologic diagnosis of ECD is often challenging due to nonspecific inflammatory and fibrotic findings on histopathologic review of tissue specimens. Additionally, the association of ECD with unusual tissue tropism and an insidious onset often results in diagnostic errors and delays. Most patients with ECD require treatment, except for a minority of patients with minimally symptomatic single-organ disease. The first ECD consensus guidelines were published in 2014 on behalf of the physicians and researchers within the Erdheim-Chester Disease Global Alliance. With the recent molecular discoveries and the approval of the first targeted therapy (vemurafenib) for BRAF-V600-mutant ECD, there is a need for updated clinical practice guidelines to optimize the diagnosis and treatment of this disease. This document presents consensus recommendations that resulted from the International Medical Symposia on ECD in 2017 and 2019. Herein, we include the guidelines for the clinical, laboratory, histologic, and radiographic evaluation of ECD patients along with treatment recommendations based on our clinical experience and review of literature in the molecular era.

Non-Coding RNAs in Normal B-Cell Development and in Mantle Cell Lymphoma: From Molecular Mechanism to Biomarker and Therapeutic Agent Potential.

B-lymphocytes are essential for an efficient immune response against a variety of pathogens. A large fraction of hematologic malignancies are of B-cell origin, suggesting that the development and activation of B cells must be tightly regulated. In recent years, differentially expressed non-coding RNAs have been identified in mantle cell lymphoma (MCL) tumor samples as opposed to their naive, normal B-cell compartment. These aberrantly expressed molecules, specifically microRNAs (miRNAs), circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs), have a role in cellular growth and survival pathways in various biological models. Here, we provide an overview of current knowledge on the role of non-coding RNAs and their relevant targets in B-cell development, activation and malignant transformation, summarizing the current understanding of the role of aberrant expression of non-coding RNAs in MCL pathobiology with perspectives for clinical use.

Ibrutinib resistance in mantle cell lymphoma: clinical, molecular and treatment aspects.

Mantle cell lymphoma (MCL) is a lymphoproliferative disorder comprising about 6-10% of all B cell lymphoma cases. Ibrutinib is an inhibitor of Bruton tyrosine kinase (BTK), a key component of early B-cell receptor (BCR) signalling pathways. Although treatment with ibrutinib has significantly improved the outcome of MCL patients, approximately one-third of the patients have primary drug resistance while others appear to develop acquired resistance. Understanding the molecular events leading to the primary and acquired resistance to ibrutinib is essential for achieving better outcomes in patients with MCL. In this review, we describe the biology of the BCR signalling pathway and summarize the landmark clinical trials that have led to the approval of ibrutinib. We review the molecular mechanisms underlying primary and acquired ibrutinib resistance as well as recent studies dealing with overcoming ibrutinib resistance.

AL amyloidosis clonal plasma cells are regulated by microRNAs and dependent on anti-apoptotic BCL2 family members.

BACKGROUND: Noncoding RNAs such as microRNAs (miRNAs) have attracted attention as biological pathway regulators, which differ from chromosomal translocations and gene point mutations. Their involvement in the molecular mechanisms underlying light chain (AL) amyloidosis pathogenesis is yet to be elucidated. AIMS: To decipher specific miRNA expression profile in AL-amyloidosis and to examine how miRNAs are involved in AL pathogenesis. METHODS: The expression profile of miRNAs and mRNA from bone marrow (BM)-derived CD138+ cells were determined using the NanoString nCounter assay and RNA-Seq, respectively. The effect of aberrantly expressed miRNAs on potential molecular targets was analyzed by qRT-PCR, Western blot, Mito-potential assay, and Annexin-PI staining. RESULTS: Genes which were significantly differentially expressed between AL-amyloidosis and MM, were found to be involved in cell growth and apoptotic mechanisms. Specifically, BCL2L1, MCL1, and BCL2 were upregulated in AL-amyloidosis compared with MM and controls. The levels of miR-181a-5p and miR-9-5p, which regulate the above-mentioned genes, were lower in BM samples from AL-amyloidosis compared with controls, providing a mechanism for BCL2 family gene upregulation. When miR-9-5p and miR-181a-5p were overexpressed in ALMC1 cells, BCL2L1, MCL1, and BCL2 were downregulated and induced apoptosis. Treatment of ALMC-1 cells with venetoclax, (BCL-2 inhibitor), resulted in the upregulation of those miRNAs, the downregulation of BCL2, MCL1, and BCL2L1 mRNA and protein levels, and subsequent apoptosis. CONCLUSION: Our findings suggest that miR-9-5p and miR-181a-5p act as tumor-suppressors whose downregulation induces anti-apoptotic mechanisms underlying the pathogenesis of AL-amyloidosis. The study highlights the post-transcriptional regulation in AL-amyloidosis and provides pathogenetic evidence for the potential use of BCL-2 inhibitors in this disease.

Mutant PIK3CA is a targetable driver alteration in histiocytic neoplasms.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm characterized by the accumulation of clonal mononuclear phagocyte system cells expressing CD1a and CD207. In the past decade, molecular profiling of LCH as well as other histiocytic neoplasms demonstrated that these diseases are driven by MAPK activating alterations, with somatic BRAFV600E mutations in >50% of patients with LCH, and clinical inhibition of MAPK signaling has demonstrated remarkable clinical efficacy. At the same time, activating alterations in kinase-encoding genes, such as PIK3CA, ALK, RET, and CSF1R, which can activate mitogenic pathways independent from the MAPK pathway, have been reported in a subset of histiocytic neoplasms with anecdotal evidence of successful targeted treatment of histiocytoses harboring driver alterations in RET, ALK, and CSF1R. However, evidence supporting the biological consequences of expression of PIK3CA mutations in hematopoietic cells has been lacking, and whether targeted inhibition of PI3K is clinically efficacious in histiocytic neoplasms is unknown. Here, we provide evidence that activating mutations in PIK3CA can drive histiocytic neoplasms in vivo using a conditional knockin mouse expressing mutant PIK3CAH1047R in monocyte/dendritic cell progenitors. In parallel, we demonstrate successful treatment of PIK3CA-mutated, multisystemic LCH using alpelisib, an inhibitor of the alpha catalytic subunit of PI3K. Alpelisib demonstrated a tolerable safety profile at a dose of 750 mg per week and clinical and metabolic complete remission in a patient with PIK3CA-mutated LCH. These data demonstrate PIK3CA as a targetable noncanonical driver of LCH and underscore the importance of mutational analysis-based personalized treatment in histiocytic neoplasms.

Overcoming Barriers to Tumor Genomic Profiling through Direct-to-Patient Outreach.

PURPOSE: To overcome barriers to genomic testing for patients with rare cancers, we initiated a program to offer free clinical tumor genomic testing worldwide to patients with select rare cancer subtypes. EXPERIMENTAL DESIGN: Patients were recruited through social media outreach and engagement with disease-specific advocacy groups, with a focus on patients with histiocytosis, germ cell tumors (GCT), and pediatric cancers. Tumors were analyzed using the MSK-IMPACT next-generation sequencing assay with the return of results to patients and their local physicians. Whole-exome recapture was performed for female patients with GCTs to define the genomic landscape of this rare cancer subtype. RESULTS: A total of 333 patients were enrolled, and tumor tissue was received for 288 (86.4%), with 250 (86.8%) having tumor DNA of sufficient quality for MSK-IMPACT testing. Eighteen patients with histiocytosis have received genomically guided therapy to date, of whom 17 (94%) have had clinical benefit with a mean treatment duration of 21.7 months (range, 6-40+). Whole-exome sequencing of ovarian GCTs identified a subset with haploid genotypes, a phenotype rarely observed in other cancer types. Actionable genomic alterations were rare in ovarian GCT (28%); however, 2 patients with ovarian GCTs with squamous transformation had high tumor mutational burden, one of whom had a complete response to pembrolizumab. CONCLUSIONS: Direct-to-patient outreach can facilitate the assembly of cohorts of rare cancers of sufficient size to define their genomic landscape. By profiling tumors in a clinical laboratory, results could be reported to patients and their local physicians to guide treatment. See related commentary by Desai and Subbiah, p. 2339.

MicroRNA-15a-5p acts as a tumor suppressor in histiocytosis by mediating CXCL10-ERK-LIN28a-let-7 axis.

Erdheim-Chester disease (ECD) is characterized by excessive production and accumulation of histiocytes within multiple tissues and organs. ECD patients harbor recurrent mutations of genes associated with the RAS/RAF/MEK/ERK signaling pathway, particularly, the BRAFV600E mutation. Following our previous finding that miR-15a-5p is the most prominently downregulated microRNA in ECD patients compared to healthy individuals, we elucidated its role in ECD pathogenesis. Bioinformatics analysis followed by a luciferase assay showed that chemokine ligand 10 (CXCL10) is a target gene regulated by miRNA-15a-5p. This was confirmed in 24/34 ECD patients that had low expression of miR-15a-5p concurrent with upregulated CXCL10. Overexpression of miR-15a-5p in cell lines harboring BRAF or RAS mutations (Ba/F3, KG-1a and OCI-AML3) resulted in CXCL10 downregulation, followed by LIN28a and p-ERK signaling downregulation and let-7 family upregulation. Overexpression of miR-15a-5p inhibited cell growth and induced apoptosis by decreasing Bcl-2 and Bcl-xl levels. Analysis of sequential samples from 7 ECD patients treated with MAPK inhibitors (vemurafenib/cobimetinib) for 4 months showed miR-15a-5p upregulation and CXCL10 downregulation. Our findings suggest that miR-15a-5p is a tumor suppressor in ECD through the CXCL10-ERK-LIN28a-let7 axis, highlighting another layer of post-transcriptional regulation in this disease. Upregulation of miR-15a-5p in ECD patients may have a potential therapeutic role.

Expedient synthesis and anticancer evaluation of dual-action 9-anilinoacridine methyl triazene chimeras.

The efficient synthesis of molecular hybrids including a DNA-intercalating 9-anilinoacridine (9-AnA) core and a methyl triazene DNA-methylating moiety is described. Nucleophilic aromatic substitution (SN Ar) and electrophilic aromatic substitution (EAS) reactions using readily accessible starting materials provide a quick entry to novel bifunctional anticancer molecules. The chimeras were evaluated for their anticancer activity. Chimera 7b presented the highest antitumor activity at low micromolar IC50 values in antiproliferative assays performed with various cancer cell lines. In comparison, compound 7b outperformed DNA-intercalating drugs like amsacrine and AHMA. Mechanistic studies of chimera 7b suggest a dual mechanism of action: methylation of the DNA-repairing protein MGMT associated with the triazene structural portion and Topo II inhibition by intercalation of the acridine core.

Dual BRAF/MEK blockade restores CNS responses in BRAF-mutant Erdheim-Chester disease patients following BRAF inhibitor monotherapy.

BACKGROUND: Erdheim-Chester disease (ECD), a rare inflammatory myeloid neoplasm, is known to be fundamentally reliant on the constitutive activation of the MAPK signaling pathway in the majority of patients. Consequently, inhibition of the V600E-mutant BRAF kinase has proven to be a safe and efficacious long-term therapeutic strategy for BRAF-mutant ECD patients. Nevertheless, in a subset of patients with CNS disease, the efficacy of long-term treatment may diminish, facilitating suboptimal responses or disease progression. METHODS: We retrospectively describe 3 BRAF-mutant ECD patients whose treatment with Vemurafenib was upgraded to Vemurafenib/Cobimetinib due to either disease progression, insufficient response, or unacceptable toxicity. CNS response to therapy was evaluated using magnetic resonance imaging (MRI) and extra-cranial disease was monitored using 18F-fludeoxyglucose positron emission tomography/computed tomography (PET/CT). RESULTS: Three patients with a mean age of 52.6 years were treated with Vemurafenib for a mean duration of 26.6 months (range: 6-52). Monotherapies were upgraded to Vemurafenib/Cobimetinib dual therapy. The combination therapy was administered for a mean duration of 21 months (range: 19-23). All patients exhibited clinical and neurological improvement. Regression of lesions on MRI was noted in 2 patients. Both patients characterized by a PET-avid disease responded to the biological treatment regimen with complete metabolic remissions. CONCLUSION: Dual inhibition of BRAF and downstream MEK may be a safe and effective therapeutic strategy for BRAF-mutant ECD patients for whom BRAF inhibitor therapy proved insufficient and as such appropriate for the long-term management of CNS disease in ECD.

The Contribution of MicroRNAs to the Inflammatory and Neoplastic Characteristics of Erdheim-Chester Disease.

The pathogenesis of histiocytic neoplasms is driven by mutations activating the MAPK/ERK pathway, but little is known about the transcriptional and post-transcriptional alterations involved in these neoplasms. We analyzed microRNA (miRNA) expression in plasma samples and tissue biopsies of Erdheim-Chester disease (ECD) and Langerhans cell histiocytosis (LCH) patients. In silico analysis revealed a potential role of miRNAs in regulating gene expression in these neoplasms as compared with healthy controls (HC). NanoString analysis revealed 101 differentially expressed plasma miRNAs in 16 ECD patients as compared with 11 HC, 95% of which were downregulated. MiRNAs-15a-5p, -15b-5p, -21-5p, -107, -221-3p, -320e, -630, and let-7 family miRNAs were further evaluated by qRT-PCR in an extended cohort of 32 ECD patients, seven LCH and 15 HC. Six miRNAs (let-7a, let-7c, miR-15a-5p, miR-15b-5p, miR-107 and miR-630) were highly expressed in LCH plasma and tissue samples as compared with ECD. Pathway enrichment analysis indicated the miRNA contribution to inflammatory and pro-survival signaling pathways. Moreover, the let-7 family members were downregulated in untreated ECD patients as compared with HC, while treatment with MAPK/ERK signaling inhibitors for 16 weeks resulted in their upregulation, which was in parallel with the radiologic response seen by PET-CT. The study highlights the potential contribution of miRNA to the inflammatory and neoplastic characteristics of ECD and LCH.

Synthesis and Biological Studies of New Multifunctional Curcumin Platforms for Anticancer Drug Delivery.

BACKGROUND: Scientists have extensively investigated curcumin, yielding many publications on treatments of cancer. Numerous derivatives of curcumin were synthesized, evaluated for their anti-oxidant and free-radical scavenging, SAR, ADME properties and tested in anticancer applications. OBJECTIVE: We decided to exploit curcumin as a bioactive core platform for carrying anticancer drugs, which likely possesses a carboxyl moiety for potential linkage to the carrier for drug delivery. METHODS: The goal of this work is to develop biolabile multifunctional curcumin platforms towards anticancer drug delivery, including determination of drug release profiling in hydrolytic media, in vitro cytotoxicity, antioxidant properties and blockage of relevant cell survival pathways. RESULTS: We report on a facile synthesis of the bioactive multifunctional curcumin-based platforms linked to a variety of anticancer drugs like amonafide and chlorambucil, and release of the drugs in a hydrolytic environment. The leading curcumin-based platform has presented antioxidant activity similar to curcumin, but with much more potent cytotoxicity in vitro in agreement with the augmented blockage of the NF-kB cell survival pathway. CONCLUSION: The approach presented here may prove beneficial for bioactive curcumin-based delivery applications where multiple drug delivery is required in a consecutive and controlled mode.

Network analysis of microRNAs, genes and their regulation in diffuse and follicular B-cell lymphomas.

MicroRNAs (miRs) are short non-coding regulatory RNAs that control gene expression at the post-transcriptional level and play an important role in cancer development and progression, acting either as oncogenes or as tumor suppressors. Identification of aberrantly expressed miRs in patients with hematological malignancies as compared to healthy individuals has suggested that these molecules may serve as novel clinical diagnostic and prognostic biomarkers. We conducted a systematic literature review of articles published between 2007 and 2017 and re-analyzed experimentally-validated human miR expression signatures in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) from various biological sources (tumor tissue, peripheral blood, bone marrow and cell lines). A unique miR expression pattern was observed for each disease. Compared to healthy individuals, 61 miRs were aberrantly expressed in DLBCL and 85 in FL; 20-30% of aberrantly expressed miRs overlapped between the two lymphoma subtypes. Analysis of integrative positive and negative miRNA-mRNA relationships using the Ingenuity Pathway Analysis (IPA) system revealed 970 miR-mRNA pairs for DLBCL and 90 for FL. Through gene ontology analysis, we found potential regulatory pathways that are deregulated in DLBCL and FL due to improper expression of miR target genes. By comparing the expression level of the aberrantly expressed miRs in DLBCL to their expression levels in other malignancies, we identified seven miRs that are aberrantly expressed in DLBCL tumor tissues (miR-15a, miR-16, miR-17, miR-106, miR-21, miR-155 and miR-34a-5p). This specific expression pattern may be a potential diagnostic tool for DLBCL.

MiR-30e induces apoptosis and sensitizes K562 cells to imatinib treatment via regulation of the BCR-ABL protein.

Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cell carrying the Philadelphia (Ph) chromosome and an oncogenic BCR-ABL fusion gene. Tyrosine kinase inhibitors (TKIs) of the BCR-ABL kinase are the treatment of choice for CML patients. Imatinib was the first TKI used in clinical practice with excellent results. MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression and play an important role in cancer development and progression. Aberrant miRNA expression profiles have been shown to be characteristic of many cancers. Here, we demonstrate that miR-30e is expressed at low levels in CML cell lines and patient samples. Bioinformatics analysis reveals a putative target site for miR-30e in the 3'-untranslated region (UTR) of the ABL gene. In agreement, luciferase assay verified that miR-30e directly targets ABL. Enforced expression of miR-30e in K562 cells suppressed proliferation and induced apoptosis of these cells and sensitized them to imatinib treatment. These findings strongly suggest that miR-30e acts as a tumor suppressor by downregulating BCR-ABL expression. Up-regulation of miR-30e in CML cells may therefore have a therapeutic efficacy against this disease.

Restoration of miR-424 suppresses BCR-ABL activity and sensitizes CML cells to imatinib treatment.

MicroRNAs (miRNAs) are small noncoding RNAs that participate in many biological processes by posttranscriptionally regulating gene expression. Dysregulation of miRNA expression has been shown to be typical of many neoplasms. Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells carrying the Philadelphia (Ph) chromosome and an oncogenic BCR-ABL tyrosine kinase fusion gene. While the development of tyrosine kinase inhibitors (TKIs) like imatinib has revolutionized treatment of CML, it has become increasingly clear in recent years that TKI treatment alone will not be curative in many cases. Thus, further dissection of the regulatory networks that drive BCR-ABL-induced malignant transformation may help to identify other novel therapeutic approaches that complement TKI treatment. In this study we demonstrate that the expression of miR-424 is markedly low in CML cell lines and patient samples at time of diagnosis. With the aid of bioinformatics analysis we revealed a conserved target site for miR-424 in the 3'-untranslated region (UTR) of the ABL gene. Via luciferase assays, we showed that miR-424 directly targets BCR-ABL. Overexpression of miR-424 was shown to suppress proliferation and induce apoptosis of K562 cells as well as sensitize these cells to imatinib treatment. These findings strongly suggest that miR-424 acts as a tumor suppressor by downregulating BCR-ABL expression. Up-regulation of miR-424 in CML cells may therefore have a therapeutic effect against this disease.

NAD(P)H quinone oxidoreductase protects TAp63gamma from proteasomal degradation and regulates TAp63gamma-dependent growth arrest.

BACKGROUND: p63 is a member of the p53 transcription factor family. p63 is expressed from two promoters resulting in proteins with opposite functions: the transcriptionally active TAp63 and the dominant-negative DeltaNp63. Similar to p53, the TAp63 isoforms induce cell cycle arrest and apoptosis. The DeltaNp63 isoforms are dominant-negative variants opposing the activities of p53, TAp63 and TAp73. To avoid unnecessary cell death accompanied by proper response to stress, the expression of the p53 family members must be tightly regulated. NAD(P)H quinone oxidoreductase (NQO1) has recently been shown to interact with and inhibit the degradation of p53. Due to the structural similarities between p53 and p63, we were interested in studying the ability of wild-type and polymorphic, inactive NQO1 to interact with and stabilize p63. We focused on TAp63gamma, as it is the most potent transcription activator and it is expected to have a role in tumor suppression. PRINCIPAL FINDINGS: We show that TAp63gamma can be degraded by the 20S proteasomes. Wild-type but not polymorphic, inactive NQO1 physically interacts with TAp63gamma, stabilizes it and protects it from this degradation. NQO1-mediated TAp63gamma stabilization was especially prominent under stress. Accordingly, we found that downregulation of NQO1 inhibits TAp63gamma-dependant p21 upregulation and TAp63gamma-induced growth arrest stimulated by doxorubicin. CONCLUSIONS/SIGNIFICANCE: Our report is the first to identify this new mechanism demonstrating a physical and functional relationship between NQO1 and the most potent p63 isoform, TAp63gamma. These findings appoint a direct role for NQO1 in the regulation of TAp63gamma expression, especially following stress and may therefore have clinical implications for tumor development and therapy.